Rolling circle amplification (RCA) involves growing a long DNA chain with a repetitive sequence by continuously adding nucleotides to a primer annealed to a circular DNA template.[1,2] The DNA polymerases used for this reaction, such as phi29 DNA polymerase, are special because they possess both strand displacement ability and high processivity.[3,4] These properties empower these enzymes to make cyclic copying of the same circular template, producing extremely long DNA molecules with thousands of sequence repeats.
RCA has emerged as a popular DNA amplification technique because it offers some key advantages that cannot be matched by polymerase chain reaction (PCR). One advantage is that it does not require equipment: while PCR needs temperature cycling, RCA is an isothermal process. No need for special equipment makes RCA better suited for point-of-care (POC) and field applications. Another advantage is the compatibility with most molecular recognition elements (MREs). Unlike PCR that requires a high-temperature (>90° C.) step that deactivates most MREs, RCA can be conducted at temperatures that are more suited for optimal MRE functions. This particular benefit facilitates the use of RCA for the detection of not only nucleic acid targets (both DNA and RNA),[5-8] but also other analytes (small molecules, proteins and even cells)[9-11] when combined with functional nucleic acid probes (e.g. aptamers and DNAzymes).[12-16] The key element in nearly all reported biosensing strategies involving RCA is linking a molecular recognition event into the formation of a primer-template complex from which the DNA polymerase synthesizes long-chain DNA amplicons. Such coupling delivers high detection sensitivity, which is crucial for diagnostic and biosensing applications.